Nucleotide sequence of a new member of the freezing tolerance-associated protein family in wheat

Exposure to low temperature induces and modulates the synthesis of specific proteins (Guy, 1990). We have previously identified a protein family specifically regulated by low temperature in wheat. Northem and westem analyses revealed that this family is composed of at least four members (Houde et al., 1992b). Characterization of the cDNA encoding the 50-kD member, Wcsl20 (Houde et al., 1992a), showed the presence of two repeated domains that were also found in dehydrins and in late embryogenesis abundant and ABAinduced protein families (Dure, 1993). A second member of this family, Wcs200, corresponding to a protein of 200 kD, has been cloned, and Southem analysis along with protein microsequencing indicated that WCS200 contains the same repeats as WCSl20 (Ouellet et al., 1993). In this report, we describe the isolation and characterization of a third member of this family (Table I). The clone encodes a protein of 66 kD that cross-reacts with the anti-WCSlZO antibody. The calculated molecular mass of the polypeptide is 46,797 D with an isoelectric point of 7.28. As is WCS120, the predicted protein is rich in Gly (26.7%), His (10.5%), and Thr (17.1%). DNA sequence comparison between Wcsl20 and Wcs66 revealed that the coding portions of these transcripts were highly homologous, with an identity of 94%, when an ‘insertion” of 234 bases is excluded (from position 406). This insertion contains one additional repeat A (GEKKGVMENIKEKLPGGHGDHQQ) and three additional repeat B (TGGTYGQQGHTGTT). Thus, the WCS66 protein possesses 7 copies of repeat A and 14 copies of repeat B. This high homology suggests that those proteins play similar roles during cold acclimation. It is likely that some of the genes from this family have been duplicated from a common ancestral gene. This gene duplication may help to allow a faster accumulation of proteins to protect cells against lowtemperature stress. Expression of these genes in the sense